This proposal is focused on cell intrinsic regulation of adult hematopoietic stem cells (HSC). The ultimate goal is to define molecular mediators of stem cell proliferation and self-renewal to enable manipulation of these for therapies, specifically therapies for blood disorders such AIDS. The prior grant period emphasized cyclin dependent kinase inhibitors (cdki), determining their role in stem cell homeostasis and how they could be manipulated to alter stem cell function. They defined in detail distinctive phenotypes associated with the genetic deficiency of p21Cip1 (p21), p27Kip1 (p27), p18INK4c (p18) and p16INK4a (p16). The phenotypes were different for each genotype, specifically in the areas of stem cell cycling and its three potential outcomes: self-renewal, differentiation and programmed cell death. This proposal will complete and build on that information, examining the molecular basis for these processes using independent, complementary genetic strategies to define how stem cells accomplish a self-renewing cell division. They will address the following specific aims: 1. Use an unbiased forward genetic screen to identify molecular participants controlling HSC self-renewal and proliferation. These experiments exploit the limited ability to maintain HSC ex vivo to select for shRNA enable expansion of HSC as demonstrated by in vivo function. 2. Identify the molecular mediators of HSC self-renewal and proliferation by comparative genetic analysis of cdki deficient mice with distinctive phenotypes 3. Validate the candidate molecular mediators of self-renewal and proliferation using in vivo analyses. Success of this project will provide both in depth understanding of key regulatory pathways for adult HSC and create targeted approaches to manipulate stem cell self-renewal for therapeutic purposes.